Obesity, and especially upper body obesity, is a common and very serious public health problem in the United States and throughout the world. According to recent statistics, more than 25% of the United States population and 27% of the Canadian population are over weight. Kuczmarski, Amer. J. of Clin. Nut. 55: 495S-502S (1992); Reeder et. al., Can. Med. Ass. J., 23:226-233 (1992). Upper body obesity is the strongest risk factor known for type II diabetes mellitus, and is a strong risk factor for cardiovascular disease and cancer as well. Recent estimates for the medical cost of obesity are $150,000,000,000 world wide. The problem has become serious enough that the surgeon general has begun an initiative to combat the ever increasing adiposity rampant in American society.
Much of this obesity induced pathology can be attributed to the strong association with dyslipidemia, hypertension, and insulin resistance. Many studies have demonstrated that reduction in obesity by diet and exercise reduces these risk factors dramatically. Unfortunately, these treatments are largely unsuccessful with a failure rate reaching 95%. This failure may be due to the fact that the condition is strongly associated with genetically inherited factors that contribute to increased appetite, preference for highly caloric foods, reduced physical activity, and increased lipogenic metabolism. This indicates that people inheriting these genetic traits are prone to becoming obese regardless of their efforts to combat the condition. Therefore, a pharmacological agent that can correct this adiposity handicap and allow the physician to successfully treat obese patients in spite of their genetic inheritance is needed.
The ob/ob mouse is a model of obesity and diabetes that is known to carry an autosomal recessive trait linked to a mutation in the sixth chromosome. Recently, Yiying Zhang and co-workers published the positional cloning of the mouse gene linked with this condition. Yiying Zhang et al. Nature 372: 425-32 (1994). This report disclosed a gene coding for a 167 amino acid protein with a 21 amino acid signal peptide that is exclusively expressed in adipose tissue.
Most recently, biologically active anti-obesity proteins are disclosed and claimed in Basinski et al., U.S. application Ser. No. 08/383,638, filed Feb. 6, 1995. These proteins are disclosed in SEQ ID NO: 1. The present invention provides a process for preparing an anti-obesity protein of SEQ ID NO: 1 using dipeptidyl-aminopeptidase isolated from the slime mold, Dictyostelium discoideum.
Dictyostelium discoideum is a primitive eukaryotic microorganism commonly called a slime mold, or more specifically, a cellular slime mold. The organism is found naturally on the surface of soil and dung. The wild type amoeba obtains nutrients exclusively by ingestion (phagocytosis) of whole bacteria; for this reason they are sometimes referred to as carnivorous. Axenic mutants of D. discoideum have been isolated that are capable of growth without coculture of "food" bacteria and therefore can be grown on soluble media.
Dipeptidylaminopeptidases (DAP) are enzymes that hydrolyze the penultimate amino terminal peptide bond releasing dipeptides from the unblocked amino-termini of peptides and proteins. There are currently four classes of dipeptidyl-aminopeptidases (designated DAP-I, DAP-II, DAP-III and DAP-IV) which are distinguished based on their physical characteristics and the rates at which they catalyze cleavage with various amino-terminal peptide sequences. DAP I is a relatively non-specific DAP that will catalyze the release of many dipeptide combinations from the unblocked amino termini of peptides and proteins. DAP I shows little or no activity if the emergent dipeptide is X-Pro, Arg-X, or Lys-X (where X is any amino acid). DAP II shows a preference for amino terminal dipeptide sequences that begin with Arg-X or Lys-X, and to a lesser extent, X-Pro. DAP-II exhibits significantly lower cleavage rates versus most other dipeptide combinations. DAP III appears to have a propensity toward amino terminal dipeptide sequences of the form Arg-Arg and Lys-Lys. DAP IV shows its highest rate of hydrolytic activity toward dipeptide sequences of the form X-Pro. The DAP enzymes, particularly DAP-I and DAP-IV, have been shown to be useful in processing proteins.
The present process employs yet another form of DAP, dDAP isolated from the cellular slime mold, Dictyostelium discoideum. Using dDAP, the anti-obesity proteins of SEQ ID NO: 1 may be prepared in high yield.